Francisella tularensis causes severe infections in animals and humans. This Gram-negative facultative intracellular bacterium is easily cultivated in vitro and the infectious dose of highly virulent strains is only 10 to 50 viable cells. F. tularensis can gain entry to the host through several portals that include broken skin, the mucosal epithelium of the eye, throat, and lungs. There is no licensed vaccine to protect against F. tularensis infections. These properties, and the fact that F. tularensis was weaponized in the past, increase the urgency towards the development of vaccines and the identification of therapeutic targets. The long-term goal of this proposal is to identify genes important for intracellular replication of F. tularensis in macrophage. Genes required for intracellular replication are likely important in model mammalian hosts and during infections in humans. Towards this goal we report the construction of a series of plasmid vectors that will allow the identification and characterization of Francisella genes contributing to intracellular survival using in vivo expression approaches. Macrophage-induced genes will be specifically knocked-out with an allelic exchange strategy in LVS and a virulent type A strain. The resulting strains will be characterized for virulence-associated phenotypes in macrophage and mouse models of infection. Characterization of newly identified in vivo expressed genes and planned purC and aroA knock-outs is anticipated to provide a selection of suitable strains for future live vaccine trials. Our macrophage reporter screen is expected to identify both structural and regulatory genes governing intracellular replication. Understanding the regulatory pathways allowing infection and multiplication is critical to deciphering the environmental cues sensed by the bacterium. The linkage of genes in regulatory cascades will be investigated utilizing microarray analyses. Overall, we propose to combine expression technology and global analyses to identify virulence determinants associated with intracellular replication in the poorly characterized select agent, F. tularensis.